Development of a multilayered palate substitute in rabbits: a histochemical ex vivo and in vivo analysis

Current tissue engineering technology focuses on developing simple tissues, whereas multilayered structures comprising several tissue types have rarely been described. We developed a highly biomimetic multilayered palate substitute with bone and oral mucosa tissues using rabbit cells and biomaterials subjected to nanotechnological techniques based on plastic compression. This novel palate substitute was autologously grafted in vivo, and histological and histochemical analyses were used to evaluate biointegration, cell function, and cell differentiation in the multilayered palate substitute. The three-dimensional structure of the multilayered palate substitute was histologically similar to control tissues, but the ex vivo level of cell and tissue differentiation were low as determined by the absence of epithelial differentiation although cytokeratins 4 and 13 were expressed. In vivo grafting was associated with greater cell differentiation, epithelial stratification, and maturation, but the expression of cytokeratins 4, 13, 5, and 19 at did not reach control tissue levels. Histochemical analysis of the oral mucosa stroma and bone detected weak signals for proteoglycans, elastic and collagen fibers, mineralization deposits and osteocalcin in the multilayered palate substitute cultured ex vivo. However, in vivo grafting was able to induce cell and tissue differentiation, although the expression levels of these components were always significantly lower than those found in controls, except for collagen in the bone layer. These results suggest that generation of a full-thickness multilayered palate substitute is achievable and that tissues become partially differentiated upon in vivo grafting.